遊離副腎細胞を用いるACTH bioassayの検討と血中ACTHの動態の観察
スポンサーリンク
概要
- 論文の詳細を見る
The determination of ACTH in plasma has become attainable through the development of the radioimmunoassay (RIA). However, the RIA method has inherent limitations, and the value obtained represents the level of immunoreactive ACTH but not necessarily the level of biologically active ACTH. Therefore, a bioassay of ACTH is still the indispensable method of investigating the pituitary-adrenal axis, physiologically and clinically. Recently Sayers et al. reported a sensitive and accurate bioassay using isolated adrenal cells (IAC). But there were many problems to be solved in the application of their technique in determining plasma ACTH concentrations.<BR>In this investigation, the preparation of IAC (which had an invariable activity), the conditions of incubation in this system, the purification of ACTH from plasma, and the protection of ACTH from its degradation were evaluated in order to improve the bioassay technique. Using this method, the dynamics of plasma ACTH at various states of the rat, dog and human were investigated.<BR>The steroidogenic response to ACTH and DBC-AMP of the cells from rats administrated with dexamethasone was more sensitive than that of cells from intact rats. By preincubating IAC, sensitivity of the cells to ACTH and DBC-AMP increased. Production of corticosteroids (CS) by the IAC from rat and dog adrenals showed the log dose response to any given quantity of ACTH. The minimum effective dose was 4 pg of <SUP>1-24</SUP>ACTH in rat IAC.<BR>IAC from rats was more sensitive to ACTH than that from dogs because CS production of dog IAC was high even when ACTH was not added.<BR>Digestion with 0.125% of trypsin brought a good recovery of freed cells from dog adrenals which responded well to ACTH. A lima bean trypsin inhibitor was more suitable for preparing IAC than was a soybean trypsin inhibitor. Some BSA contained ACTH activi-ty, which was able to be excluded with QUSO treatment.<BR>The steroidogenic response of IAC was suppressed when incubation was performed with unextracted plasma. It was necessary to extract ACTH from plasma. The use of QUSO absorption of plasma ACTH, with a subsequent aqueous acetone elution and evaporation of the eluate at a high temperature was successful in removing interfering substances. ACTH determination with IAC was carried out using the beaker in which the QUSO eluate was evaporated at 60-70°C. By this extraction procedure, no interfering effects were noted as far as the recovery of a small amount of ACTH was examined in 10 human plasma samples.<BR>Plasma ACTH was stable for 3-5 hours when venepuncture was performed with the addition of Trasylol or EDTA and the blood was stored at 4°C.<BR>Inter-assay (CV : 4.3-17.7%) or intra-assay (9.4-19%) reproducibilities and recovery (87-117%) of this assay system were satisfactory.<BR>With this assay system, it was possible to detect the resting levels of ACTH of the rat, dog and human using 2-4ml of plasma.<BR>Following the administration of acetylcholine in dogs, a good correlation was observed between the height of the peak plasma ACTH and the adrenal 11-OHCS output during 60 minutes. The disappearance rates of endogenous ACTH and exogenous ACTH (porcine ACTH or <SUP>1-24</SUP> ACTH) in dogs were 2.8, 2.3 and 1.5 minutes, respectively.<BR>Plasma ACTH concentrations in normal human subjects were 18-169pg/ml at 6 : 00 and 0-78pg/ml at 18 : 00 respectively. The administration of metyrapone or stress during abdominal surgery was followed by an increase in plasma ACTH concentrations. Plasma ACTH concentrations in untreated patient with Addison's disease, ectopic ACTH producing tumors, and adrenogenital syndrome were 2480, 2000 and 430pg/ml, respectively.<BR>Sensitivity, precision and specificity of this asssay system using IAC are excellent, and this bioassay system is applicable for the determination of biologically active ACTH levels in plasma.
- 日本内分泌学会の論文